Lyme disease is the most prevalent tick-borne disease in the United States. Borrelia burgdorferi (Bb), the causative agent, is highly motile and can traverse complex environments inside mammalian and arthropod hosts during its infectious cycle. The central hypothesis of this application is that the motility and chemotaxis of Bb constitute a unique paradigm and play a pivotal role in the host-vector cycle as well as in the disease process, e.g., dissemination, tissue tropism, and immune evasion. During our last funding period, several unique aspects about Bb motility and chemotaxis were unveiled. In collaboration with several other groups, we have pushed Bb motility research to the forefront, and now Bb has emerged as a paradigm for in-depth understanding of spirochete motility and chemotaxis. In this renewal application, a comprehensive study will be carried out to elucidate the molecular mechanisms that account for the unique aspects that we have observed as well as their links to Bb pathogenicity. Bb has 7-11 internally localized periplasmic flagella (PFs) nea the cell poles. They form a distinct ribbon wrapping around the cell cylinder, which have both skeletal and motility functions. This unique arrangement and function have not been reported in any other flagellated bacteria. Aim 1 will define the molecular mechanism that controls the number and placement of PFs and determine how this control mechanism affects the virulence of Bb. As an enzootic pathogen, Bb has two different chemosensory pathways. It has been hypothesized that these two pathways function in different hosts during the infectious cycle, e.g., one in mammals and the other in ticks. Aim 2 will decipher the unique aspect of chemotaxis and its role in the enzootic cycle of Bb. Bb is highly motile and outruns (>10-fold faster) host immune cells in mouse dermis. We hypothesize that this ability allows Bb to rapidly disseminate through dense skin tissue ahead of the cellular immune responses and then to disseminate to distal organs in mammals. Aim 3 will test our hypothesis by analyzing two motility and chemotaxis mutants in mice using recently developed live-imaging techniques. All of the studies proposed in this renewal are novel and have not been carried out in any spirochetes. Completion of these studies will lead to a new level of molecular analysis of Bb motility and chemotaxis as well as an understanding of their precise roles in the pathogenesis of Lyme disease. In addition, the fundamental knowledge to be gained here is high-impact and could aid in the understanding of the unique motility in other pathogenic spirochetes.